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Chromosome number and genome size diversity in five genera Citation: A. Teixeira Mesquita, M.V. Romero-da Cruz, A.L. Sousa Azevedo, E.R. Forni-Martins (2019) Chromo- some number and genome size diver- Amanda Teixeira Mesquita1,*, Marìa Victoria Romero-da Cruz1, Ana sity in five Solanaceae genera. Caryo- Luisa Sousa Azevedo2, Eliana Regina Forni-Martins1 logia 72(3): 105-115. doi: 10.13128/ caryologia-772 1 Departamento de Biologia Vegetal, Universidade Estadual de Campinas, Rua Monteiro Lobato 255, CEP: 13.083-970 Campinas (SP), Brasil Published: December 13, 2019 2 Embrapa Gado de Leite, Empresa Brasileira de Pesquisa Agropecuária (Embrapa), Rua Copyright: © 2019 A. Teixeira Mesqui- Eugênio do Nascimento 610, CEP: 36.038-330 Juiz de Fora (MG), Brasil ta, M.V. Romero da Cruz, A.L. Sousa *Corresponding author: [email protected] Azevedo, E.R. Forni Martins. This is an open access, peer-reviewed article published by Firenze University Press Abstract. Sixteen species of Solanaceae, belonging to five genera, were studied karyo- (http://www.fupress.com/caryologia) logically through chromosome counting, chromosomal measurement, and karyotype and distributed under the terms of the symmetry. Genome size (GS) estimation was performed on fifteen species using flow Creative Commons Attribution License, cytometry. The chromosome number 2n=24 was found in all Solanum species and which permits unrestricted use, distri- Acnistus arborescens, 2n=22 was found in Brunfelsia uniflora, and 2n=16 in bution, and reproduction in any medi- representatives. pubescens was the only specie with evidence of polyploidy, um, provided the original author and showing 2n=4x=48 chromosomes. The chromosome numbers of S. adspersum, S. inodo- source are credited. rum, S. flaccidum, S. sanctae-catharinae, and B. uniflora were reported for the first time. Data Availability Statement: All rel- Haploid karyotype length (HKL) was statistically different between the studied species. evant data are within the paper and its The polyploid P. pubescens showed the largest HKL value, 93.10 µm. In general, kary- Supporting Information files. otypes were symmetrical with predominance of metacentric chromosomes. Chromo- some size was small in most species (<4 µm), while S. diploconos, C. laevigatum, and Competing Interests: The Author(s) C. mariquitense, species with high HKL values, exhibited larger chromosomes. Genome declare(s) no conflict of interest. size estimation were unpublished for ten studied species and were the first estimation for the genera Acnistus, Brunfelsia and Physalis. Were observed about eight-fold differ- ences between species with averages varying from 2C=2.57 pg to 2C=20.27 pg. As both HKL and GS showed a continuous variation. We observed partial similarity in the spe- cies ordered according to HKL and GS. The Solanaceae genera showed a constant chro- mosome number and a tendency to posse symmetrical karyotypes. The genome size also showed differences, which suggests that chromosome evolution in the group could be driven by alterations in the repetitive fractions of the genome.

Keywords. Acnistus, Brunfelsia, Cestrum, Physalis, Solanum, karyotype evolution.

INTRODUCTION

The Solanaceae family comprises about 2,500 species and 100 genera and have cosmopolitan distribution. The greatest diversity of the family is found in Neotropical regions (D’Arcy 1991; Hunziker 2001). Members of Solanace- ae have great ecological and morphological diversity, characteristics which favoured the occupation of diverse habitats, such as desert regions, tropi-

Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(3): 105-115, 2019 ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-772 106 Amanda Teixeira Mesquita et al. cal rainforests, and even disturbed areas (D’Arcy 1991; Only about 8% of Solanaceae taxa have available GS Knapp 2002). data. This character has more variability than chromo- The family includes several species of important some number (Soltis et al. 2003). In Solanum, the GS global food crops with high economic value, such as ranges of from forty-fold in species with 2n=24 chromo- tomatoes (Solanum lycopersicum), potatoes (Solanum somes. The smallest reported C-value is in S. chacoense, tuberosum), eggplants (Solanum melongena), and chilli 1C=0.63 pg (Bennett and Smith 1976), while the largest peppers (Capsicum spp.), widely used drug , such value is 1C= 24.80 pg, found in S. hartwegii (Pringle and as tobacco (Nicotiana tabacum), “datura” (Datura stra- Murray 1991). monium), and “angel’s tears” Brugmansia suaveolens, as Nevertheless, there are still many gaps in karyotypic well as many ornamental plants, such as species of the knowledge for the Solanaceae family and such informa- genus Brunfelsia, Cestrum and Petunia. Many Solan- tion (i.e. genome size, chromosome number, and kar- aceae species, including tomatoes, potatoes, and tobacco, yotype variables) is important to complete current data are model organisms for various biological studies, and and to better understand the systematic relationships their genomes are some of the most well studied among and chromosome evolution of the family. Therefore, the angiosperms (Knapp et al. 2004). objectives of this study were: (1) to report original chro- Karyotype information about species and groups mosome numbers and describe the karyotype variables are important for taxonomic and evolutionary stud- in distinct genera of the Solanaceae family, (2) to deter- ies, whereas karyological changes accompany specia- mine the genome size (GS) using flow cytometry for the tion and, consequently, the diversification of the groups first time for many species. (Guerra et al. 2008, 2012, Chiarini et al. 2018). The chro- mosome number, nuclear DNA content, total length of the chromosome complement, asymmetry indices, and MATERIAL AND METHODS number and location of the rDNA sites and heterochro- matic bands are the main data used in cytotaxonomic material studies. Chromosome number data is the most available information and is not influenced by external agents, Sixteen species from the genera Acnistus, Brunfelsia, such as age of individuals, environmental conditions, Cestrum, Solanum, and Physalis were collected in South- and gene expression, providing accurate data about spe- eastern Brazil. Voucher specimens were deposited into cies evolution (Dobginy et al. 2004, Guerra et al. 2008, the Herbarium at the University of Campinas (UEC). 2012). Cytogenetic characterization, accompanied by a Data collection is detailed in Table 1. genome size (GS) study, can offer important informa- tion about genome organization, phylogenetic relation- Chromosome preparations ships, and evolutionary trends. This approach has been successful used in some Solanaceae (Mishiba et al. 2000, Seeds of at least three individuals per species were Moscone 2003, Chiarini et al. 2014). germinated in Petri dishes. In some cases, 1 ml gibberel- Chromosome data is available for some genera of lic acid (GA3) was applied to break seed dormancy (Ellis Solanaceae, while for other genera there is not enough et al. 1985). According to previous tests, root meristems data or information about their chromosomes. Lycium were pre-treated with different solutions to block the cell and Solanum present constant chromosome number cycle to obtain good chromosome spread and condensa- (2n=24 and polyploids) (Bernadello and Anderson 1990; tion (Table 2). The root apices were fixed in 3:1 ethanol: Bernadello et al. 1994; Chiarini and Bernadello 2006; acetic acid (v:v) mixture that was stirred for a minimum Rego et al. 2009; Stiefkens et al. 2010; Melo et al. 2011; of 12 h at room temperature (RT) and stored at -6ºC Chiarini et al. 2014), while Capsicum shows 2n=24 and until slide preparation. Slides were made using root mer- 2n=26 (Moscone 1993; Moscone et al. 2007; Aguilera et istems that were previously digested in a solution of 1% al. 2014; Grabiele et al. 2014; Romero-da Cruz and For- macerozima, 2% cellulase, and 20% pectinase for 10-15 ni-Martins 2015; Romero-da Cruz et al. 2017). For the minutes at 37ºC and squashed in a drop of 45% acetic Cestreae tribe, composed of Cestrum, Sessea, and Vestia, acid. Coverslips were removed after freezing in liquid the only chromosome number reported to date is 2n=16 nitrogen for 15 minutes. The cells were photographed (Fregonezi et al. 2006; Las Peñas et al. 2006; Fernandes under a microscope Olympus BX51 with a DP72 camera et al. 2009; Urdampilleta et al. 2015). The greatest range attached and images were captured using Olympus DP2 in chromosome number is found in Nicotiana (n=12 to BSW program (Olympus Corporation). n=32, and polyploids, Chase et al. 2003). Chromosome number and genome size diversity in five Solanaceae genera 107

Table 1. Cytogenetics data of Solanaceae species: Species and voucher specimen; provenance of materials; chromosome number, haploid karyotype formula (HKF), median haploid karyotype length (HKL), variation in chromosome length (VCL); symmetry indices A1 and A2; median DNA content (2C values).

HKL – µm 2C values – pg Species (Voucher specimen) Provenance 2n HKF VCL – µm A1 A2 (CI) (CI) Acnistus arborescens Schltdl. Brazil: Rio Grande do Sul; Aratinga 24 12m 45.93 (2.78) 3.17-4.38 0.17 0.10 6.56 (0.06) (Monge 2787) Brunfelsia. uniflora D. Don Brazil; São Paulo; Campinas 22 7m+4sm 50.51 (0.50) 3.89-5.37 0.32 0.10 6.58 (0.13) (Mesquita 15) Cestrum laevigatum Schltdl. Brazil; São Paulo; Campinas 16 6m+2sm 78.72 (2.96) 7.92-10.88 0.23 0.10 20.27 (0.43) (Mesquita 12) C. mariquitense Kunth (Mesquita Brazil; São Paulo; Campinas 16 7m+1sm 73.91 (6.38) 7.35-11.39 0.21 0.12 - 14) Physalis pubescens L. (Vasconcellos Brazil; São Paulo; Jundiaí 48 19m+5sm 93.10 (2.87) 1.43-2.80 0.32 0.18 12.98 (0.09) Neto 00-068) Solanum Cyphomandra clade Solanum diploconos (Mart.) Bohs Brazil; São Paulo; Jundiaí 24 8m+4sm 74.72 (1.86) 4.63-7.49 0.32 0.14 19.22 (0.43) (Mesquita 23) Dulcamaroid clade S. flaccidum Vell. (Mesquita 07) Brazil; São Paulo; Campinas 24 9m+2sm+1st 26.73 (1.14) 1.83-2.50 0.27 0.10 2.57 (0.25) S. inodorum Vell. (Vasconcellos Brazil; São Paulo; Jundiaí 24 5m+7sm 38.33 (3.90) 2.83-3.86 0.39 0.09 4.63 (0.06) Neto 20401) Geminata clade S. pseudocapsicum L. (Mesquita 24) Brazil; São Paulo; Jundiaí 24 9m+3sm 28.61 (7.70) 1.76-2.72 0.28 0.13 2.94 (0.11) Leptostemonum clade Acanthophora section S. acerifolium Sendt. (Mesquita 02) Brazil; São Paulo; Campinas 24 10m+2sm 36.17 (1.09) 1.71-3.87 0.26 0.23 5.69 (0.15) S. palinacanthum Dunal (Mesquita Brazil; São Paulo; Ubatuba 24 5m+7sm 37.86 (0.72) 2.51-3.86 0.41 0.13 5.00 (0.10) 20) Torva section S. adspersum Witasek (Monge 2748 Brazil; Rio de Janeiro; Arraial do 24 9m+3sm 25.09 (1.62) 1.77-2.44 0.25 0.09 3.19 (0.04) c 240) Cabo S. scuticum M. Nee (Vasconcellos Brazil; São Paulo; Jundiaí 24 9m+3sm 27.66 (2.10) 1.95-2.79 0.31 0.04 3.42 (0.06) Neto 8503) S. variabile Mart (Monge 2324) Brazil; São Paulo; Itacaré 24 9m+3sm 33.45 (0.51) 2.15-3.26 0.24 0.11 3.54 (0.09) Uncertain position S. concinnum Schott ex Sendtn. Brazil; São Paulo; Campinas 24 6m+6sm 31.82 (0.37) 2.26-2.86 0.39 0.09 3.65 (0.25) (Mesquita 08) S. sanctae-catharinae Dunal Brazil; São Paulo; Jundiaí 24 10m+2sm 23.15(2.56) 1.68-2.35 0.26 0.09 3.79 (0.07) (Vasconcellos Neto 20873)

CI – Confidence interval at 95% of semi range.

Karyotype analysis type length (HKL) was calculated by the sum of the hap- loid chromosome lengths. The arm ratio (r) was calcu- Five metaphases of each species, with the same lated using the formula r= L/S and was used to classify degree of chromosome condensation, were used to deter- chromosomes according to Levan et al. (1964). For ideo- mine the chromosome number. The measurements were grams, chromosomes were first grouped by morphology taken using the MicroMeasure© software (3.3). Ideo- (r=1.00-1.69 metacentric-m; r=1.70-2.99 submetacentric- grams were made using measurements of the following sm; r=3.00-6.99 subtelocentric-sm) and then by decreas- means for each chromosome pair: S (short arm length), ing size order within each group. L (long arm length) and C (total chromosome length) The karyotype symmetry was described using the using the formula C= S+L. In addition, haploid karyo- indices A1= 1-[(Σbi/Bi)/n] (bi = mean of the short arm 108 Amanda Teixeira Mesquita et al. of each chromosome pair, Bi = average of the long arm RESULTS of each chromosome pair, n = number of chromosome pairs) and A2=x/s (s = standard deviation; x = average Karyotype analysis chromosome complement length) (Zarco 1986). A1 index measures intrachromosomal asymmetry which indicates The somatic chromosome numbers were 2n=2x=24 differences in the size of chromosome arms. A2 index (Acnistus and Solanum), 2n=2x=22 (Brunfelsia), 2n=2x=16 measures the interchromosomal asymmetry and indi- (Cestrum) and 2n=4x=48 (Physalis) (Table 1; Fig. 1). cates the variation in chromosome lengths. In terms of Although the differences in HKL between some length, chromosomes were classified according to Lima species were significant (p<0.05) according to statisti- de Faria (1980) as very small (≤1 µm), small (>1 µm and cal analysis (Table 1; Fig. 2), this variation was gradual, ≤4 µm), intermediate (>4 and ≤12) and big (>12 and ≤60). and no groups were formed. Solanum sanctae-catharinae showed the lowest median value (23.15 µm) with a vari- ation of 1.68-2.35 µm from the smallest to largest chro- Flow cytometry mosome pair. Other Solanum species also presented low HKL (except for S. diploconos), with values reach- The same species that were cytogenetically analysed ing 38.33 µm in S. inodorum (2.83 to 3.86 µm). Species (except for Cestrum mariquitense) were cultivated in a with intermediate HKL values were A. arborescens (45.93 greenhouse and used for GS measurements. For each µm, 3.17 to 4.38 µm) and B. uniflora (50.51 µm, 3.89 to species, three individuals were measured in three repeti- 5.38 µm). High HKL values were found in C. mariq- tions, for a total of nine samples. Approximately 1 cm2 of uitense with 73.91 µm (7.35 to 11.39 µm), S. diploconos young leaf tissue was used to prepare the nuclear suspen- with 74.72 µm (4.63 to 7.49 µm), and C. laevigatum with sions, according to Dolezel et al. (2007). The material of 78.72 µm (7.92 to 10.88 µm). Physalis pubescens showed each species of interest and a piece of internal leaf stand- ard (Pisum sativum “Ctirad” 2C=9.09 pg ( Dolezel et al. 1998) were sliced with a razor blade and placed into a Petri dish on ice. About 1 ml of LB01 buffer (Dolezel et al. 1989) was used to extract the nuclei. A nylon mesh with 40 microns was used to filter the sample (CellTrics, PARTEC), then, 25 µL 1 mg/mL propidium iodide and 25 µL 1 mg/mL RNAse were added to the nuclear suspen- sion. The measurement was performed on a BD FACS Calibur flow cytometer, for each sample an average of 10,000 nuclei were analysed. The 2C value was calculated using the linear relationship between fluorescence signals from stained nuclei of the unknown sample and the ref- erence standard. The nomenclature for genome size clas- sification followed Leitch et al. (1998) with modification by Soltis et al. (2003): values <1.4 pg and between 1.4 to 3.5 pg correspond to “very small” and “small” genomes, respectively. On the other hand, values between 3.51- 13.99 pg, >14 pg and >35 pg are considered “intermedi- ate,” “large”, and “very large” genomes, respectively.

Statistical analyses

The HKL values, as well of GS values of each species, were compared using Past 3.18 ® (Øyvind Hammer, Nat- ural History Museum, University of Oslo). The Kruskal- Wallis nonparametric test was performed to compare the averages among the species and Dunn’s post-hoc test Fig. 1. Somatic metaphases of five genera of Solanaceae.a Solanum (Dunn 1954) was carried out after significant Kruskal- flaccidum. b S. adspersum. c S. sanctae-catharinae. d S. inodorum. e Wallis test. Physalis pubescens. f Acnistus arborescens. g Brunfelsia uniflora. h S. diploconos. i Cestrum laevigatum. Bar=10 µm. Chromosome number and genome size diversity in five Solanaceae genera 109

cies are shown in Fig. 4. According to statistical analy- sis, GS showed significant differences among some of the studied species (Fig. 5). A variation of about eight-fold was observed, ranging from 2C=2.57 pg (S. flaccidum, Fig. 4a) to 2C=20.27 pg (C. laevigatum, Fig. 4d). The GS presented continuous variation, so distinct groups were not characterized (Fig. 5). Most species had small (2C=2.57 pg in S. flacidum to 2n=3.79 pg in S. sanctae- catharinae) and intermediate genomes (2C=4.63 pg in S. inodorum to 6.56 pg in A. arborescens and 6.58 pg in B. uniflora). The species with larger genomes were P. pube- scens (2n=12.98 pg), S. diploconos (2C=19.22 pg), and C. Fig. 2. Boxplots illustrating the continuous variability of HKL laevigatum (2C=20.27 pg). (Haploid Karyotype Length), as inferred from de Kruskal Wallis analysis. The numbers on the x axis represent the species ordered by crescent HKL values (in µm): S. sanctae-catharinae (1), S. adspersum (2), S. flaccidum (3), S. scuticum (4), S. pseudocapsicum DISCUSSION (5), S. concinnum (6), S. variabile (7) S. acerifoium (8) S. palinacan- thum (9) S. inodorum (10), A. arborescens (11) B. uniflora (12) C. Chromosome number mariquitense (13) S. diploconos (14) C. laevigatum (15) P. pubescens (16). The central box represents 50% of the data from de upper to The chromosome number data found here are new lower quartile. The horizontal bar expresses the median position. The extremity of the vertical lines indicates minimum and maxi- for S. adspersum, S. inodorum, S. flaccidum, and S. sanc- mum values of HKL, if they are no outliners. When outliners are tae-catharinae, with 2n=24 chromosomes, as well as for present, they are represented by circles. B. uniflora, with 2n=22. For the remaining species, the chromosome number obtained corroborated with data found in the literature for Acnistus (2n=24), Cestrum the highest HKL value (93.10 µm), even though it is a (2n=16), Solanum (2n=24), and Physalis (2n=48) (Heiser polyploid species with chromosomes ranging from 1.43 1963; Pedrosa et al. 1999; Fernandes et al. 2009; Rego et to 2.8 µm. al. 2009; Urdampilleta et al. 2015). Karyotypes are symmetrical with A1 and A2 values All the species in this study, except for P. pubescens, for each species ranging from 0.17 to 0.41 and from 0.04 which is a tetraploid, are diploid. Although diploid is the to 0.23, respectively. Most species presented a predomi- most frequent ploidy level (including other species of nance of metacentric chromosomes (Table 1, Fig. 3) that Physalis), polyploidization has played an important role characterized most intrachromosomal symmetry shown in the evolution of some Solanaceae genera (e.g., Nico- in the A1 index. Acnistus arborescens had the most sym- tiana, Chase et al. 2003; S. elaeagnifolium, Scaldaferro et metrical karyotype, composed of only metacentric chro- al. 2012). The chromosome number most frequent in the mosomes and A1=0.17. Three species had less symmet- family is 2n=24, found in more than 85% of the previ- rical karyotypes: Solanum inodorum and S. palinacan- ously studied Solanaceae species (Olmstead et al. 2008) thum showed predominance of submetacentric chromo- though a diploid series from 2n=14 to 2n=26 is present somes (5m+7sm) and A1 value of 0.39 and 0.41, respec- in some genera (eg. Petunia and Calibrachoa, Mishiba tively. Solanum concinnum also presented A1=0.39, but et al. 2000, Cestrum, Sessea and Vestia, Las Peñas et al. karyotype formulae 6m+6sm. 2006, Capsicum, Moscone et al. 2007). Interchromosomal index A2 showed that all spe- Many authors have postulated hypotheses for the cies have few variations in chromosome size of the kar- ancestral chromosome base number in the family. yotypes. Solanum scuticum showed the small A2 value Raven (1975) proposed x=7 and 12 for the order Sola- (0.04) and Solanum acerifolium presented the highest A2 nales and Solanaceae family, respectively, while Badr value (0.23), characterizing the most interchromosomal et al. (1997) suggested the hypothesis of x=7 or x=8. asymmetry among studied species (Table 1). Moscone (1992) corroborate with the proposition of Badr et al. (1997), suggested x=7 as the basic chromo- some number for Solanaceae. Olmstead and Palmer C-value (1992) and Olmstead et al (2008) based in phylogenetic studies, suggests an ancestral position of subfam. Ces- Genome size estimates of all the studied species troideae (x=8), and x=12 as a derivate basic chromo- are shown in Table 1 and histograms for selected spe- some number the family. 110 Amanda Teixeira Mesquita et al.

Fig. 3. Ideograms of the investigated Solanaceae species based on median chromosome values. Bar=5 µm. Chromosome number and genome size diversity in five Solanaceae genera 111

Fig. 5. Boxplots illustrating the continuous variability of GS (Genome Size), as inferred from de Kruskal Wallis analysis. The numbers on the x axis represent the species ordered by crescent C-values (in pg): S. flaccidum (1), S. pseudocapsicum (2), S. adsper- sum (3), S. scuticum (4), S. variabile (5), S. concinnum (6), S. sanctae- catharinae (7) S. inodorum (8) S. palinacanthum (9) S. acerifolium Fig. 4. Flow cytometry histograms (iodide propidium fluorescence (10), A. arborescens (11) B. uniflora (12) P. pubescens (13) S. diplo- intensity of nuclei) showing DNA amounts from leaf tissues of conos (14) C. laevigatum (15). The central box represents 50% of the some Solanaceae species. 1 Pisum sativum “Ctirad” (standard). 2 S. data from de upper to lower quartile. The horizontal bar expresses flaccidum. 3 P. pubescens. 4 A. arborescens. 5 C. laevigatum. the median position. The extremity of the vertical lines indicates minimum and maximum values of HKL, if they are no outliners. When outliners are present, they are represented by circles. The lack of chromosomal data for several genera and for the Solanaceae sister group, the family Convolvu- laceae, as well as the presence of distinct basic numbers ered a species of the distinct genus Cyphomandra), and in other families, as in Hydroleaceae, x=8 and P. pubescens, have been reported in some studies for 10 (Constance 1963) and Montiniaceae, x=12 (Goldblatt Solanum (Bernardello and Anderson 1990; Acosta et al. 1979), has hampered to establish a consensus about a 2005; Chiarini et al. 2006; Rego et al. 2009; Melo et al. basic chromosome number and understand the direction 2011; Moyetta et al. 2013), and another Solanaceae gen- of chromosome number evolution for the family. era, as Lycianthes and Vassobia (Rego et al. 2009) and Lycium, (Stiefkens and Bernadello 2002,) Acnistus arborescens and B. uniflora shows chromo- Karyotype structure somes and consequently, HKL values, with intermedi- ate size, when compared to Solanum and Physalis. These Differences in chromosome size were seen between karyotype characteristics are also present in Capsicum the Solanaceae species here investigated. (Moscone 1996), Sclerophylax and Nolana (Lujea and The relatively small chromosome size and HKL Chiarini 2017), genera also belonging to Solanaceae. observed in the species here of Solanum, except for S. Although the intermediate size of the chromosomes, a diploconos (statistically distinct, and previously consid- constant chromosome number, karyotype symmetry and chromosomes majority metacentric appear to maintain in these groups. Table 2. Pretreatments used for each genus of Solanaceae studied. The tribe Cestreae (subfam. Cestroideae) embrac- es the genera Cestrum, Sessea and Vestia, presents the Genus Pretreatments largest chromosomal sizes of the family (Fregonezi et 8-hydroxyquinoline 0.002M + al. 2006, Peñas et al. 2006). Cestrum laevigatum and C. Acnistus and Physalis cycloheximide 25mg/L (1:1), 8 h, 4ºC mariquitense, investigated here, showed the largest chro- Brunfelsia 8-hydroxyquinoline 0.002M, 6 h, 14ºC mosome size high HKL values, confirming the trend for Cestrum Colchicine 0.1% 6 h, RT* the tr. Cestreae (Fregonezi et al. 2006). Such increase in Saturated solution of r-dichlorobenzene 2 Solanum chromosome size for the tribe can be due to the absence h, RT* of Arabidopsis-type telomeres (TTTAGGG)n for short Saturated solution of r-dichlorobenzene 5 Solanum (S. diploconos) interstitial telomeric sequences (SITS), leading to the h, RT* lack of control of the telomerase-dependent replication. *RT=room temperature. These sequences may associate with other DNA sequenc- 112 Amanda Teixeira Mesquita et al. es and assist in their dispersion leading to an increase in evolutionary history of Solanaceae, thereby preserving genome size (Sykorova et al. 2003a, b). chromosome morphology favouring chromosomal uni- Besides chromosome number, other widely con- formity served karyotype characters in Solanaceae genera, are chromosome morphology and karyotype symmetry. Symmetrical karyotypes with a predominance of meta- C-value/DNA content centric chromosome pairs are found in the five genera studied here. Acnistus arborescens was the unique spe- Despite the great number of representatives in Sola- cie that had only metacentric chromosomes, thus, had naceae, the GS estimation is available for a small propor- the most symmetrical karyotype and only S. flaccidum tion of species and genera. Only 12 Solanaceae genera showed a subtelocentric chromosome pair. Other gen- have data about GS, representing approximately 10% era of the family Solanaceae in which it is possible to and 186 species that corresponding to 7% of Solanaceae observe these characteristics areCapsicum (Pozzobon et representatives. Genome size data for A. arborescens, B. al. 2006; Moscone et al. 1993, 2007), Lycium (Stiefkens uniflora and P. pubescens are the first estimation for the and Bernadello 2012), Lycianthes, and Vassobia (Rego et relative genera. Some species of Cestrum and Solanum al. 2009). have their GS measured but the data here obtained are Among the angiosperms, karyotype asymmetry can unpublished for C. laevigatum, S. flaccidum, S. inodor- be associated with derivate taxa (Stebbins 1971) In some um, S, adspersum, S, scuticum, S. variabile, S. concinnum groups of the Solanaceae family, intermediate asymme- and S. sanctae-catharinae. try values, can be observed, such as the tr. Cestreae (Las The GS variation observed in the species studied Peñas et al. 2006), Solanum sect. Acanthophora (Chiarini partially coincides with the variation observed in HKL. et al. 2014) However, for species, the karytotype asym- In general, species with small, intermediary, or high metry was not associate with basal or derived position of HKL presented the same GS classification. Among the the taxa in the phylogeny. Regarding karyotype asym- five species with small HKL, four presented small values metry, no evolutive trend was found for the analysed of DNA content (S. adspersum, S. flaccidum, S. pseudo- genera or among the representatives of Solanum. Over- capsicum and S. scuticum). Similarly, of the six species all, karyotype asymmetry seems to occur randomly with high HKL, five showed high values of DNA content within some groups of the family. (A. arborescens, B. uniflora, S. diploconos, C. laeviga- Our study analysed five species from other sections tum and P. pubescens). The estimation of nuclear DNA (Acantophora and Torva) of the Leptostemonum clade. using flow cytometry is more accurate than measuring In sect. Acantophora (S. acerifolium and S. palinacan- chromosomes. This accuracy is supported by statistical thum), we observed greater HKL and karyotype asym- tests and by species boxplots, where the dispersion of metry than in sect. Torva (S. adspersum, S. scuticum HKL data (Figure 2) was greater than GS data (Figure and S. variabile). Karyotype asymmetry was previously 5). Calculating HKL is more subject to external effects reported for the Lepstotemomun clade (Chiarini et al. (Stace 2000). Methodological standardization, especially 2014). In other cases, asymmetry was random within the degree of chromosomal condensation in the mitotic a group, as in Solanum Morelloid and Dulcamaroid metaphase, is important for obtaining chromosomal siz- clades (Moyetta et al. 2013). Both species belonging to es and comparing the results obtained between species clade Dulcamaroid that were studied corroborated this (Stace 2000). data, while S. inodorum (HKL=38.33 µm) presented a Although angiosperms have high diversity in their more asymmetrical karyotype (5m + 7sm and A1=0.39, DNA content, the predominance of a small genome size A2=0.09) than S. flaccidum (HKL=26.73 µm, 9m + 2sm causes a tendency with modal values equal to 1C = 0.7 + 1st and A1=0.27, A2=0.10). pg (Leitch et al. 1998). This distribution, strongly skewed The karyotype characteristics described for the stud- towards small genomes, is associated with the ancestral ied species and genera as well as in other Solanaceae condition of the group and large genomes could have groups, a constancy in the chromosome number, karyo- arose more than once during angiosperm evolution type symmetry and chromosome morphology, indicates (Leitch et al. 1998; Soltis et al. 2003). karyotype orthoselection, which preserves similar chro- Among the species studied, and in the Solanaceae mosomal complements, regardless of chromosome size family in general, we observed a predominance of small (Acosta et al. 2005; Moscone et al. 2003). According to genomes (see Bennett and Leitch 2012). 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